Deciphering the relationship between animal behavior and cellular activity in the central nervous system (CNS) is perhaps one of the greatest challenges in neuroscience research today. Traditionally, electrophysiological approaches have been used to sparsely sample from electrically excitable cells of freely moving animals. This has led to the discovery of important behaviorally related phenomena such as place, grid, and head-direction cells in the brain and central pattern generator (CPG) neurons in the spinal cord. Optical imaging in combination with new labeling approaches now allows minimally invasive and comprehensive sampling from dense networks of electrically and chemically excitable cells, such as neurons and glial cells. Imaging in head- restrained mobile mice and with miniaturized head-borne microscopes, for example, has led to the discovery of unanticipated forms of behaviorally related neuronal and glial cell excitation in cortical and hippocampal microcircuits. Long wavelength two- and three-photon excitation now enables imaging in brain regions previously accessible only by invasive endoscopic methods. In contrast, imaging in the spinal cord, the primary neurological link between the brain and other parts of the body, is limited to superficial dorsal regions in anesthetized animals. Because anesthesia precludes animal behavior and alters cellular activity, and because essential central pattern generator components are located in deep tissue regions key aspects of spinal cord physiology have remained elusive. Additionally, because current imaging approaches are limited to either the spinal cord or brain, little is known about how the communication between these CNS regions contributes to behavior. Overcoming such critical barriers in the study of CNS function and dysfunction requires development and application of new tools and approaches. As part of this application new tools and approaches for minimally invasive optical recordings from spinal cord microcircuits during animal behavior, from presently inaccessible deep spinal cord regions, and from anatomically connected brain-spinal cord networks will be developed. The rationale for the proposed research is that once these barriers have been overcome new and unanticipated insight into spinal cord physiology and pathology will be gained. Three specific aims will be pursued: 1) Enable study of spinal cord microcircuits in behaving mice through development of restraint and freely moving imaging approaches; 2) Enable minimally invasive study of deep spinal cord regions in live mice through development of adaptive infrared imaging approaches; and 3) Enable minimally invasive study of spinal cord-brain communication in live mice through development of parallel imaging approaches. Together, the proposed research contribution is significant because it will provide new and unanticipated insight into how defined cell types and their activity patterns relate to spinal cord physiology, brain-spinal cord communication, and animal behavior. It is innovative because it will provide a unique set of tools and approaches with groundbreaking possibilities in multiple areas of science.